The long term goal of this research is to develop SERS/SERRS as a general technique for the study of biological systems. The enormous sensitivity provided by the enhanced Raman effect has considerable potential for fundamental studies and also for bioanalytical applications. During the initial phase of this work procedures for obtaining SERS/SERRS spectra for a variety of biological molecules were developed which preserve the native structure. The applicability of this technique for structure/functional analysis of water soluble and membrane bound (photosynthetic and gap junction) proteins, nucleic acids and model systems was successfully demonstrated. During the proposed funding period the previously developed SERS technique, together with electrochemical methods, visible, Uv-RR and time resolved Raman spectroscopy, will be used to study a problem of fundamental interest and to develop a new analytical technique. The two specific aims to be addressed include the following: (1) SERRS, resonance and UV resonance Raman spectroscopy, together with voltammetric measurements will be applied to a wide variety of cytochrome c and cytochrome c3 mutants in order to characterize the protein/heme interactions and their effect on the electron transfer properties. The combination of SERRS with electrochemical methods will be used to follow the structural changes in the heme which occur during electron transfer process. UV-RR scattering will provide a probe of the aromatic amino acid residues in different mutants. Time resolved R&an spectroscopy will be utilized to measure the kinetics of electron transfer between the cytochromes and their natural redox partners in solution and at the electrode surface using the SERRS technique. Chemically modified cytochrome c and covalently linked cytochrome c/plastocyanin complexes will be studied as well. (2) SERRS-Linked Immunoassay (SLI) is proposed as a new, highly sensitive and highly specific method for detecting biologically important molecules. A universal SLI procedure which can be used for different clinical and environmental analyses will be developed during the proposed funding period. Two types of model systems will be examined in order to validate this method: paraquat as an example of small molecules with environmental importance and ferritin as an example of a large molecule for clinical application. The goal will be to reach detection limits in the attomolar range.